Bottom Line:
As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term.Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes.The fluorescent nanodiamond technology is able to overcome the limitations of previously used organic fluorophores, thus appearing as a choice methodology for studying distribution, persistence and long-term neurotoxicity of alum adjuvants and beyond of other types of nanoparticles.

Background: Aluminum oxyhydroxide (alum) is a crystalline compound widely used as an immunologic adjuvant of vaccines. Concerns linked to alum particles have emerged following recognition of their causative role in the so-called macrophagic myofasciitis (MMF) lesion in patients with myalgic encephalomyelitis, revealing an unexpectedly long-lasting biopersistence of alum within immune cells and a fundamental misconception of its biodisposition. Evidence that aluminum-coated particles phagocytozed in the injected muscle and its draining lymph nodes can disseminate within phagocytes throughout the body and slowly accumulate in the brain further suggested that alum safety should be evaluated in the long term. However, lack of specific staining makes difficult the assessment of low quantities of bona fide alum adjuvant particles in tissues.

Methods: We explored the feasibility of using fluorescent functionalized nanodiamonds (mfNDs) as a permanent label of alum (Alhydrogel(®)). mfNDs have a specific and perfectly photostable fluorescence based on the presence within the diamond lattice of nitrogen-vacancy centers (NV centers). As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term. We thus developed fluorescent nanodiamonds functionalized by hyperbranched polyglycerol (mfNDs) allowing good coupling and stability of alum:mfNDs (AluDia) complexes. Specificities of AluDia complexes were comparable to the whole reference vaccine (anti-hepatitis B vaccine) in terms of particle size and zeta potential.

Results: In vivo, AluDia injection was followed by prompt phagocytosis and AluDia particles remained easily detectable by the specific signal of the fND particles in the injected muscle, draining lymph nodes, spleen, liver and brain. In vitro, mfNDs had low toxicity on THP-1 cells and AluDia showed cell toxicity similar to alum alone. Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes.

Conclusions: The fluorescent nanodiamond technology is able to overcome the limitations of previously used organic fluorophores, thus appearing as a choice methodology for studying distribution, persistence and long-term neurotoxicity of alum adjuvants and beyond of other types of nanoparticles.

Mentions:
At 21 days after i.m. injection, AluDia particles accumulated into the injected muscle similarly to vaccine particles [3]. Indeed, granuloma mainly composed of CD11b+ monocyte- macrophage lineage cells filled with AluDia was formed in the endomysium, i.e. in between myofibers, at the injection site (Fig. 2a). Non Morin-stained AluDia particles in muscles have the same fluorescent signature as compared to those of mfNDs (Fig. 2b,c). These particles do not display any fluorescence when they are excited at 420nm as compared to Morin-stained AluDia (Fig. 2c). The phase contrast image shows AluDia particles within the granuloma region in muscle section (Fig. 2d). Morin stain for aluminum confirmed that macrophages contained stably associated AluDia particles as assessed by both red and green fluorescence (Fig. 2e-g). Importantly, photostability of mfNDs upon long laser exposure made AluDia detection very easy without background fluorescence whereas the detection of Morin stain was commonly disturbed by its bleaching and a strong tissue fluorescent background (Fig. 2h-j). Serial sectioning of the injected muscle at day 45, day 135, day 180 and day 270 after AluDia injection showed progressive shrinkage of muscle granulomas (Table 2), as previously reported in rats [30]. At 270 days post-injection, one out of three tested mice was completely free of muscle granuloma, and the other two mice only had small residual muscle granulomas.Fig. 2

Mentions:
At 21 days after i.m. injection, AluDia particles accumulated into the injected muscle similarly to vaccine particles [3]. Indeed, granuloma mainly composed of CD11b+ monocyte- macrophage lineage cells filled with AluDia was formed in the endomysium, i.e. in between myofibers, at the injection site (Fig. 2a). Non Morin-stained AluDia particles in muscles have the same fluorescent signature as compared to those of mfNDs (Fig. 2b,c). These particles do not display any fluorescence when they are excited at 420nm as compared to Morin-stained AluDia (Fig. 2c). The phase contrast image shows AluDia particles within the granuloma region in muscle section (Fig. 2d). Morin stain for aluminum confirmed that macrophages contained stably associated AluDia particles as assessed by both red and green fluorescence (Fig. 2e-g). Importantly, photostability of mfNDs upon long laser exposure made AluDia detection very easy without background fluorescence whereas the detection of Morin stain was commonly disturbed by its bleaching and a strong tissue fluorescent background (Fig. 2h-j). Serial sectioning of the injected muscle at day 45, day 135, day 180 and day 270 after AluDia injection showed progressive shrinkage of muscle granulomas (Table 2), as previously reported in rats [30]. At 270 days post-injection, one out of three tested mice was completely free of muscle granuloma, and the other two mice only had small residual muscle granulomas.Fig. 2

Bottom Line:
As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term.Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes.The fluorescent nanodiamond technology is able to overcome the limitations of previously used organic fluorophores, thus appearing as a choice methodology for studying distribution, persistence and long-term neurotoxicity of alum adjuvants and beyond of other types of nanoparticles.

Background: Aluminum oxyhydroxide (alum) is a crystalline compound widely used as an immunologic adjuvant of vaccines. Concerns linked to alum particles have emerged following recognition of their causative role in the so-called macrophagic myofasciitis (MMF) lesion in patients with myalgic encephalomyelitis, revealing an unexpectedly long-lasting biopersistence of alum within immune cells and a fundamental misconception of its biodisposition. Evidence that aluminum-coated particles phagocytozed in the injected muscle and its draining lymph nodes can disseminate within phagocytes throughout the body and slowly accumulate in the brain further suggested that alum safety should be evaluated in the long term. However, lack of specific staining makes difficult the assessment of low quantities of bona fide alum adjuvant particles in tissues.

Methods: We explored the feasibility of using fluorescent functionalized nanodiamonds (mfNDs) as a permanent label of alum (Alhydrogel(®)). mfNDs have a specific and perfectly photostable fluorescence based on the presence within the diamond lattice of nitrogen-vacancy centers (NV centers). As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term. We thus developed fluorescent nanodiamonds functionalized by hyperbranched polyglycerol (mfNDs) allowing good coupling and stability of alum:mfNDs (AluDia) complexes. Specificities of AluDia complexes were comparable to the whole reference vaccine (anti-hepatitis B vaccine) in terms of particle size and zeta potential.

Results: In vivo, AluDia injection was followed by prompt phagocytosis and AluDia particles remained easily detectable by the specific signal of the fND particles in the injected muscle, draining lymph nodes, spleen, liver and brain. In vitro, mfNDs had low toxicity on THP-1 cells and AluDia showed cell toxicity similar to alum alone. Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes.

Conclusions: The fluorescent nanodiamond technology is able to overcome the limitations of previously used organic fluorophores, thus appearing as a choice methodology for studying distribution, persistence and long-term neurotoxicity of alum adjuvants and beyond of other types of nanoparticles.